Female Non-obese Diabetic Research Articles

Overexpression of the reg gene in non-obese diabetic mouse pancreas during active diabetogenesis is restricted to exocrine tissue.

We demonstrated pancreatic reg gene overexpression in non-obese diabetic (NOD) mice during active diabetogenesis. The aim of this study was to determine in which part of the pancreas (endocrine and/or exocrine) the gene(s) and the protein(s) were expressed and if their localization changed with progression of the disease. In situ hybridization analysis and immunocytochemical studies were carried out on pancreas of female and male NOD mice. Both develop insulitis but diabetes develops only in females and in males only when treated by cyclophosphamide. Our results show that whatever the age, sex, and presence of insulitis and/or diabetes, the expression of reg mRNAs and of the corresponding protein(s) was restricted to exocrine tissue. Moreover, reg remains localized in acinar cells in the two opposite situations of (a) cyclophosphamide-treated males in a prediabetic stage presenting a high level of both insulin and reg mRNAs, and (b) the overtly diabetic females with no insulin but a high level of reg mRNA. These findings suggest that overexpression of the reg gene(s) might represent a defense of the acinar cell against pancreatic aggression.

Islet Abnormalities Associated with an Early Influx of Dendritic Cells and Macrophages in NOD and NODscid Mice

In the nonobese diabetic (NOD) mouse model for type 1 diabetes, the inflammatory infiltration of islets starts with an influx of dendritic cells (DC) and macrophages (Mφ) at approximately 4 weeks of age. Around this time, NOD mice show endocrine abnormalities, indicated by a transient hyperinsulinemia that lasts until 8 weeks of age. Subsequently, they develop abnormally large islets of Langerhans, here designated as “mega-islets.” NODscid mice, which lack functional lymphocytes, also exhibit transient hyperinsulinemia, but to a lesser extent. First, to determine the role of lymphocytes in the morphological islet abnormalities, we compared 6-week-old (prediabetic) NOD and NODscid females regarding mega-islet development and accumulation of antigen-presenting cells (APC), particularly CD11c+ DC and ERMP23+ Mφ. In NODscid mice, early APC infiltration and mega-islets were present, but less marked compared with NOD mice, thus suggesting a role of lymphocytes in mega-islet formation. In both NOD and NODscid mice, the APC infiltration was predominantly found around the mega-islets, suggesting a relationship between both parameters. Second, to analyze the role of β-cell hyperactivity in mega-islet formation, we studied the effect of short-term prophylactic insulin treatment on these parameters. Prophylactic insulin treatment decreased the percentages of mega-islets in both NOD and NODscid mice, indicating that β-cell hyperactivity is also involved in mega-islet formation. In conclusion, mega-islet formation in mice with the NOD genetic background takes place under the influence of both β-cell hyperactivity and leukocytes.

Preventive effect of Ninjin-to (Ren-Shen-Tang), a Kampo (Japanese traditional) formulation, on spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice.

We previously found that ingestion of an extract of Ninjin-to (NJT; Ren-Shen-Tang) suppressed the development of autoimmune diabetes in C57BL/KsJ mice induced by multiple low doses of streptozotocin. To verify this effects on spontaneous autoimmune diabetes, the effects of NJT on NOD mice were investigated in the present study. NJT, provided in drinking water (0.25%, 450 mg/kg/day) from 6 weeks of age, significantly prevented the incidence of spontaneous diabetes in female NOD mice at 30 weeks of age (2/10) compared with that of the controls (7/10), with no effects on body growth or food intake. Even in non-diabetic mice, the blood glucose levels of the NOD controls gradually increased with age, while such increase in NJT-treated mice was significantly suppressed by preventing any deficiency of glucose tolerance. NJT also significantly suppressed the progression of insulitis, which causes insulin deficiency and diabetes. It is well known that NOD mice develop insulitis and diabetes because of their Th1-dominant autoimmune response. IFN-gamma production from splenic T lymphocytes stimulated with anti-CD3 monoclonal antibodies was increased, whereas IL-4 production was decreased in NOD controls compared to age- and sex-matched normal ICR mice. NJT-treatment reduced these deviations of cytokine production in NOD mice. These data all suggest that NJT can prevent spontaneous insulitis and diabetes by the modification of deviated cytokine production in NOD mice.

Intradermal Administration of GAD & Evaluation of Diabetes Incidence in Mice: Possible Relevance for Skin Tests in Humans

In vitro cell mediated reactivity to Glutamic Acid Decarboxylase (GAD) has been reported in man and in the non-obese diabetic (NOD) mouse. The demonstration of such reactivity in vivo using GAD in a simple intradermal skin test would be useful for mass screening of subjects at risk of Type 1 diabetes. Such a skin test could be simply applied to the forearm, then signs of local reaction would indicate patients at risk. However, in order to safely apply a skin test of this type it must be certain that administration of the antigen does not itself provoke or start the process leading to diabetes in susceptible individuals. In the present study the NOD mouse model was used. GAD and two peptides of GAD, which may have relevance to the disease process, were applied intradermally to these mice to determine whether a local reaction could be seen and to see if the diabetes rate was altered. Moreover, Balb/c mice, which can be considered to be at zero risk of developing the disease, were also injected with the same GAD and GAD peptides. No significant differences were seen in the diabetes incidence of the treatment groups compared to the control groups in either the NOD or Balb/c mice although a local swelling was seen in female NOD mice susceptible to diabetes after GAD administration in the footpad. We conclude that the administration of GAD and/or GAD peptides does not provoke or accelerate diabetes incidence in the NOD mouse and that an intradermal skin-test with GAD may be suitable for preliminary trials aimed at large scale screening of humans for their potential to develop type 1 diabetes

Changes in the growth hormone-IGF-I axis in non-obese diabetic mice.

We investigated the changes in GH-IGF-I axis in non-obese diabetic (NOD)-mice, a model of insulin dependent diabetes mellitus. Diabetic female NOD mice and their age- and sex-matched controls were sacrificed at 4, 14, 21 and 30 days (30d DM) after the onset of glycosuria. Serum GH levels increased and serum IGF-I levels decreased in the 30d DM group (182 ± 32% and 45 ± 24% of age-matched controls respectively, p < 0.05). Another group (30d DM + I) was given SC insulin, and its serum IGF-I levels remained decreased. Liver GH receptor (GHR) and GH binding protein (GHBP) mRNA levels, as well as liver membrane GH binding assays were deeply decreased in the 30d DM group in comparison to controls. GHR message and binding capacity remained decreased in the 30d DM + I group. Renal GHR mRNA was decreased at 21d DM but not at 14d DM, whereas GHBP mRNA remained unchanged throughout the experiment. In conclusion, increased serum GH levels are documented in NOD diabetic mice, similarly to the changes described in humans. The decrease in GHR levels and decreased serum IGF-I in spite of increased circulating GH suggest a state of GH resistance.

Interleukin-13 prevents autoimmune diabetes in NOD mice.

Interleukin (IL)-13 is a cytokine primarily produced by the T-helper (Th)-2 subset of lymphocytes that possesses powerful anti-inflammatory properties. Here, we have evaluated the impact of IL-13 treatment on development of type 1 diabetes in diabetes-prone nonobese diabetic (NOD) mice. Prolonged treatment with recombinant human IL-13 (hIL-13) markedly diminished the incidence of spontaneous type 1 diabetes in the mice. Female NOD mice treated from age 5-16 weeks with hIL-13 also showed significantly milder insulitis than control mice. The preventive action of hIL-13 was associated with a slight but significant change from a type 1 to a type 2 cytokine response. Accordingly, splenic lymphoid cells (SLC) from hIL-13-treated mice secreted less interferon (IFN)-gamma upon ex vivo stimulation with Concanavalin A than controls, and anti-CD3 monoclonal antibody-induced activation of T-cells in vivo resulted in lower blood levels of IFN-gamma and tumor necrosis factor-alpha and augmented blood levels of IL-4 in NOD mice pretreated with hIL-13. hIL-13 treatment also increased the blood levels of IgE and inhibited the transfer of type 1 diabetes by spleen cells from a diabetic donor to irradiated recipients. Taken together, these data add hIL-13 to the list of cytokines capable of downregulating immunoinflammatory diabetogenic pathways in NOD mice, and further support the concept that IL-4-related anti-inflammatory cytokines might have a role in the prevention of type 1 diabetes.

Nicotinamide decreases MHC class II but not MHC class I expression and increases intercellular adhesion molecule-1 structures in non-obese diabetic mouse pancreas.

Pancreases of untreated and nicotinamide (NIC)-treated pre-diabetic (10-week-old) and overtly diabetic (25-week-old) female NOD (non-obese diabetic) mice and of NON (non-obese non-diabetic) control mice were studied, with the following results. (1) Islets and ducts of overtly diabetic untreated NOD mice (25-week-old) were found to express low levels of MHC class I and II molecules, like NON controls, and high levels of adhesive molecules. (2) NIC was able to slightly affect glycaemia and insulitis, slowing down diabetes progression. Moreover it significantly decreased MHC class II expression (but not class I) in vivo by week 10, and significantly enhanced intercellular adhesion molecule-1 (ICAM-1) expression, mainly by week 25, within the pancreas, where 5-bromo-2'-deoxyuridine positive nuclei and insulin positive cells were present, demonstrating that a stimulation of endocrine cell proliferation occurs. (3) In addition, NIC partly counteracted the fall of superoxide dismutase levels, observed in untreated diabetic NOD animals. (4) In vitro studies demonstrated that NIC: (i) was able to significantly reduce nitrite accumulation and to increase NAD+NADH content significantly, and (ii) was able to increase the levels of interleukin-4, a T helper 2 lymphocyte (Th2) protective cytokine, and of interferon-alpha (IFN-alpha), which is known to be able to induce MHC class I and ICAM-1 but not MHC class II expression, as well as IFN-gamma, which is also known to be able to induce MHC class I and ICAM-1 expression. The latter, although known to be a proinflammatory Th1 cytokine, has also recently been found to exert an anti-diabetogenic role. This study therefore clearly shows that adhesive mechanisms are ongoing during the later periods of diabetes in pancreatic ducts of NOD mice, and suggests they may be involved in a persistence of the immune mechanisms of recognition, adhesion and cytolysis and/or endocrine regeneration or differentiation processes, as both NIC-increased ICAM-1 expression and 5-bromo-2'-deoxyuridine positivity imply. The effects of NIC on MHC class II (i.e. a reduction) but not class I, and, mainly, on ICAM-1 expression (i.e. an increase), together with the increase in Th2 protective cytokine levels are very interesting, and could help to explain its mechanism of action and the reasons for alternate success or failure in protecting against type 1 diabetes development.

Effects of BCG, lymphotoxin and bee venom on insulitis and development of IDDM in non-obese diabetic mice.

To investigate whether BCG, lymphtoxin (LT) or bee venom (BV) can prevent insulitis and development of diabetes in non-obese diabetic (NOD) mice, we measured the degree of insulitis and incidence of diabetes in 24 ICR and 96 female NOD mice. NOD mice were randomly assigned to control, BCG-, LT-, and BV-treated groups. The BCG was given once at 6 weeks of age, and LT was given in 3 weekly doses from the age of 4 to 10 weeks. The BV was injected in 2 weekly doses from the age of 4 to 10 weeks. Diabetes started in control group at 18 weeks of age, in BCG group at 24 weeks of age, and in LT- or BV-treated group at 23 weeks of age. Cumulative incidences of diabetes at 25 weeks of age in control, BCG-, LT-, and BV-treated NOD mice are 58, 17, 25, and 21%, respectively. Incidence and severity of insulitis were reduced by BCG, LT and BV treatment. In conclusion, these results suggest that BCG, LT or BV treatment in NOD mice at early age inhibit insulitis, onset and cumulative incidence of diabetes.

Disorganization of Thymic Medulla Precedes Evolution Towards Diabetes in Female NOD Mice

The thymic medulla is a complex microenvironment which plays a crucial role in central tolerance induction. Using a quantitative histological analysis of non-obese diabetic (NOD) mice, we show that the medulla undergoes several structural modifications during the course of the disease in NOD mice. Indeed, the majority of 70-day-old NOD mice show a scattering of medullary epithelial cells in the cortex which is associated with a reduction in the size of the medulla in heavily disorganized thymuses. The severity of this phenotype is shown to correlate with the subsequent appearance of diabetes in older female NOD mice. This trait is mainly controlled by non-major histocompatibility complex NOD genes since C57BL/6 H-2g7 congenic mice have a normal medulla. It persists in conditions where effector lymphocytes that lead to diabetes are inhibited in periphery. These results suggest that primary alterations of the thymic stroma might play a role in the progression towards diabetes in NOD mice.

An immunohistochemical study of macrophage influx and the co-localization of inducible nitric oxide synthase in the pancreas of non-obese diabetic (NOD) mice during disease acceleration with cyclophosphamide.

Cyclophosphamide has been used to accelerate and synchronize diabetes in non-obese diabetic (NOD) mice. It was injected to 70-day-old female NOD mice and its effect on the progression of insulitis studied at days 0, 4, 7, 11 and at onset of diabetes. Pancreatic sections were also examined for the influx of CD4 and CD8 T cells and macrophages following immunofluorescence staining. The kinetics of macrophage immunoreactive cells in the exocrine and intra-islet areas were also investigated. Light and confocal microscopy were-employed to examine the expression and co-localization of inducible nitric oxide synthase following dual- and triple-label immunofluorescence histochemistry. After cyclophosphamide administration, the severity of insulitis remained similar from days 0 to 4 but began to rise at day 7 and markedly by day 11 and at onset of diabetes. At these two later stages, the insulitis scores were close to 100% while in age-matched control groups the insulitis scores were considerably lower. Immunohistochemical staining showed increasing numbers of CD4 and CD8 T cell subsets and macrophages within the islets and in exocrine, sinusoidal and peri-vascular regions. At onset of diabetes, several islets contained prominent clusters of macrophage immunoreactive cells. Macrophage influx into the islets increased sharply from day 7 (mean number per islet: 119 +/- 54 SEM), peaked at day 11 (mean number per islet: 228 +/- 42), and then declined at onset of diabetes (mean number per islet: 148 +/- 49). Several cells with immunolabelling for inducible nitric oxide synthase were detectable from day 7 onwards until the onset of diabetes. Dual- and triple-label immunohistochemistry showed that a significant proportion of macrophages and only a few beta cells contained the enzyme. Macrophages positive for the enzyme were located as clusters or occasionally contiguously, in the peri-islet and intra-islet areas but rarely in the exocrine region. Islets with minimal distribution of macrophages in the peri-islet areas were not positive for inducible nitric oxide synthase. Beta cells positive for the enzyme were observed in islets with significant macrophage infiltration in locations close to macrophages. The present results show that cyclophosphamide administration to female NOD mice results in a rapid influx of CD4 and CD8 cells and macrophages. The marked up-regulation of inducible nitric oxide synthase in a selective proportion of macrophages, within the islets, immediately preceding and during the onset of diabetes suggests that nitric oxide released by islet macrophages may be an important molecular mediator of beta cell destruction in this accelerated model of insulin-dependent diabetes mellitus.

Thymus-dependent monoclonal antibody-induced protection from transferred diabetes.

It is well established that long-term protection from insulin-dependent diabetes mellitus (IDDM) can be afforded to non-obese diabetic (NOD) mice by a short course of non-depleting (nd) anti-CD4 monoclonal antibodies (mAb). Since it is increasingly apparent that the CD8+ T cell plays a prominent role in the development of IDDM, we have investigated the effect of an anti-CD8 mAb (YTS 105) of the same isotype in both spontaneous and induced IDDM in NOD mice. Treatment with YTS 105 for 3 weeks was able to prevent the transfer of IDDM for a long period, and also substantially reduced spontaneous IDDM in female NOD mice. The role of the thymus in tolerance induction by these antibodies was studied. In the adult transfer model, thymectomized NOD mice, unlike their euthymic counterparts, were not protected long-term by treatment with YTS 105, and began to become overtly diabetic shortly after treatment. This was also true when the nd anti-CD4 mAb was used. Protection from spontaneous disease was not affected in the same way by thymectomy. The reasons for the observed effect of the thymus in the transfer model, and the differences between the two models that may explain the contrasting results are discussed.

Thymus-dependent monoclonal antibody-induced protection from transferred diabetes

It is well established that long-term protection from insulin-dependent diabetes mellitus (IDDM) can be afforded to non-obese diabetic (NOD) mice by a short course of non-depleting (nd) anti-CD4 monoclonal antibodies (mAb). Since it is increasingly apparent that the CD8+ T cell plays a prominent role in the development of IDDM, we have investigated the effect of an anti-CD8 mAb (YTS 105) of the same isotype in both spontaneous and induced IDDM in NOD mice. Treatment with YTS 105 for 3 weeks was able to prevent the transfer of IDDM for a long period, and also substantially reduced spontaneous IDDM in female NOD mice. The role of the thymus in tolerance induction by these antibodies was studied. In the adult transfer model, thymectomized NOD mice, unlike their euthymic counterparts, were not protected long-term by treatment with YTS 105, and began to become overtly diabetic shortly after treatment. This was also true when the nd anti-CD4 mAb was used. Protection from spontaneous disease was not affected in the same way by thymectomy. The reasons for the observed effect of the thymus in the transfer model, and the differences between the two models that may explain the contrasting results are discussed.

Islet expression of perforin, Fas/Apo-1 and interleukin-1 converting enzyme (ICE) in non-obese diabetic (NOD) mice

The aim of the present study was to correlate the islet expression of the apoptosis-associated factors Fas/Apo-1, FasL, ICE and perforin with the progression of β-cell destruction in non-obese diabetic (NOD) mice. For this purpose, thymus and isolated pancreatic islets from male and female NOD mice of 5 and 15 weeks of age were subjected to immunoblot analysis. Islet expression of the Fas/Apo-1 receptor and ICE were increased in islets from female mice 15 weeks of age as compared to corresponding males. No Fas/Apo-1 or ICE signal was observed in the 5-week-old mice. The expression of perforin increased both in islets and in thymus with age and female gender. Islet expression of FasL could not be detected. Culture of isolated islets from NMRI mice in the presence of interleukin-1 β (IL-1 β) induced the expression of ICE. However, addition of Fas-activating antibody, either alone or in combination with IL-1 β, to isolated islets did not induce DNA fragmentation. The present results support a direct role of the Fas/FasL and the perforin systems in the autoimmune destruction of insulin producing cells.

Loss of Glucose Transporter-2 Precedes Insulin Loss in the Nonobese Diabetic and the Low-Dose Streptozotocin Mouse Models: A Comparative Immunohistochemical Study by Light and Confocal Microscopy

Glucose transporter-2 (glut2) is underexpressed in β cells of several rodent models of non-insulin-dependent diabetes mellitus (NIDDM). This may also be true for rodent models of insulin-dependent diabetes mellitus (IDDM). The present study examines two murine models of autoimmune IDDM, the nonobese diabetic (NOD) and the low-dose streptozotocin (stz) murine models for changes in the expression of glut2 by double-label light and confocal microscopy during various stages of the disease. The spatial distribution of glut2 cells was also examined in relation to insulin immunoreactive cells and the islet inflammatory cells during these stages. In both the female NOD mouse and the female Swiss mouse without stz treatment, glut2 colocalized with insulin in virtually all the β cells. In the NOD mouse, islets with moderate to advanced insulitis showed either an absence or considerably reduced expression of glut2 in insulin-containing β cells. Cells with reduced glut2 expression were usually located adjacent to the region of insulitis. At onset of diabetes, glut2 immunolabeling was reduced despite the preservation of weak insulin immunoreactivity. In Swiss mice treated repeatedly with stz, glut2 labeling began to decline in selected β cells after the fourth injection in approximately 50% of the islets, despite the lack of insulitis. At this stage expression of glut2 fell in a small number of islets with evidence of early macrophage infiltration. Loss of glut2 became more pronounced in nondiabetic Swiss mice after the fifth injection. At this stage glut2 labeling in the plasma membrane appeared diffuse and variable. At onset of stz-induced diabetes, glut2 expression significantly fell, despite weak immunoreactivity for insulin. This loss was associated with an enhanced influx of both macrophages and T lymphocytes within the islets of diabetic mice. In both the NOD and the low-dose stz mouse models, loss of glut2 thus occurs from an early stage and precedes hyperglycaemia. This loss may be mediated by immune and nonimmune mechanisms.

Effects of interleukin-12 in vitro on pancreatic islets isolated from normal rodents and from non-obese diabetic mice.

Administration of the T-helper 1 (Th 1) cell promoting cytokine interleukin-12 (IL-12) accelerates the development of autoimmune diabetes in non-obese diabetic (NOD) mice. In this study we examined the effects of IL-12 on isolated islets from NMRI (Naval Medical Research Institute-established) mice, Sprague-Dawley (S-D) rats and NOD mice. NMRI and S-D islets were cultured in medium RPMI 1640 + 10% fetal calf serum and exposed for 48 h to recombinant mouse IL-12 (0, 0.1, 1 and 10 ng/ml). Islet glucose metabolism, as measured by glucose oxidation rate, was suppressed by about 25% in NMRI islets exposed to 10 ng/ml IL-12. In rat islets 0.1 ng/ml IL-12 induced a 20% decrease in glucose oxidation rate. Islets cultured with 10 ng/ml IL-12 showed a decrease in medium insulin accumulation both in mouse and rat. Glucose-stimulated insulin release was lowered in rat islets exposed to 10 ng/ml IL-12, but not affected in NMRI islets. In NMRI islets IL-12 did not influence nitric oxide production as measured by nitrite formation. In rat islets IL-12 induced a decrease in nitrite formation compared with control islets. Islets were isolated from female NOD mice (age 5, 12, 20 and 26 weeks) and examined either immediately or cultured for 7 days with 10 ng/ml IL-12 alone or in combination with 4 ng/ml of the T-cell stimulating cytokine interleukin-2 (IL-2). In the age groups > 5 weeks of age the glucose-stimulated insulin release was lower in freshly isolated compared with cultured control islets. IL-2 + IL-12 addition induced a small decrease in glucose-stimulated insulin release in islets from 12-week-old animals. With increasing age the DNA content in freshly isolated islets increased due to immune cell infiltration. The DNA content in cultured islets was decreased by 40-60% compared with freshly isolated islets in the age groups over 5 weeks. Islet insulin content was similar in both freshly isolated and cultured islets. None of the cytokines, either alone or in combination, affected islet DNA or insulin content. We conclude that IL-12 has minor suppressive effects in vitro on normal rodent islets. It is likely that the reported accelerated diabetes development of IL-12 administration to NOD mice in vivo is not mediated by a direct toxic effect to the islets. The suppressed insulin release in NOD mouse islets treated with IL-2 + IL-12 suggests, however, that the accelerating effect might partly be attributed to stimulation of immune cells present in the insulitic lesion.

Melatonin Rhythms in Mice: Role in Autoimmune and Lymphoproliferative Diseases

Abstract: Production of melatonin (MLT) in the pineal gland (PG) of inbred mice such as C57BI/6J, BALB/c, and AKR strains is still a matter of debate. In a recent study we validated the presence of MLT in the PG of these inbred mice. We found a short‐term MLT peak in the middle of the dark period with a pattern that mirrors that found previously in the serum. In another study, based on the known immunoregulatory role of MLT, we investigated the role of the PG and MLT in autoimmune diabetes mellitus type I using, as an experimental model, female nonobese diabetic (NOD) mice. Mice were pinealectomized or treated chronically with MLT (injected subcutaneously or administered via drinking water). We found that neonatal pinealectomy accelerates the development of disease in female NOD mice, whereas exogenous MLT protects animals. This is in spite of the fact that MLT increased the production of insulin autoantibodies (IAA). We conclude that PG and MLT influence the development of autoimmune diabetes, although the mechanism of action needs further investigation.

Immunosuppression preventing concordant xenogeneic islet graft rejection is not sufficient to prevent recurrence of autoimmune diabetes in nonobese diabetic mice.

We and others have reported previously that the immunosuppressant, leflunomide (Lef), can prevent allogeneic and xenogeneic islet graft rejection in streptozocin (STZ)-induced diabetic animals. However, whether Lef required to prevent islet graft rejection is sufficient to prevent the recurrence of autoimmune diabetes has not been addressed. The effect of Lef on concordant xenogeneic islet graft in STZ-induced diabetic mice and autoimmune nonobese diabetic (NOD) mice were studied. Then, whether Lef prevents the onset of spontaneous diabetes in young NOD mice and the recurrence of diabetes after major histocompatibility complex (MHC)-matched islet transplantation in diabetic NOD mice were investigated. In STZ-induced diabetic BALB/c mice, Lef treatment significantly prolonged rat islet graft survival. However, Lef could not significantly prolong rat islet graft survival in autoimmune diabetic NOD mice. For prevention studies, treatment with Lef at 30 mg/ kg/day from 4 weeks to 20 weeks of age significantly reduced the incidence of spontaneous diabetes in NOD mice. However, when the NOD mice were treated from 8 to 24 weeks of age, the incidence of spontaneous diabetes was not significantly reduced as compared to the incidence of diabetes in the untreated female NOD mice at 28 weeks of age. Finally, in the MHC-matched islet transplant model, Lef could not significantly prolong MHC-matched nonobese diabetes-resistant mice islet graft survival in NOD mice. Lef preventing concordant xenogeneic islet graft rejection is not sufficient to prevent the recurrence of autoimmune diabetes in NOD mice. We believe that controlling autoimmunity after islet transplantation will lead the way to promote successful clinical islet transplantation in the future.

Alpha/beta-T cell receptor (TCR)+CD4-CD8- (NKT) thymocytes prevent insulin-dependent diabetes mellitus in nonobese diabetic (NOD)/Lt mice by the influence of interleukin (IL)-4 and/or IL-10.

We have previously shown that nonobese diabetic (NOD) mice are selectively deficient in alpha/beta-T cell receptor (TCR)+CD4-CD8- NKT cells, a defect that may contribute to their susceptibility to the spontaneous development of insulin-dependent diabetes mellitus (IDDM). The role of NKT cells in protection from IDDM in NOD mice was studied by the infusion of thymocyte subsets into young female NOD mice. A single intravenous injection of 10(6) CD4-/lowCD8- or CD4-CD8- thymocytes from female (BALB/c x NOD)F1 donors protected intact NOD mice from the spontaneous onset of clinical IDDM. Insulitis was still present in some recipient mice, although the cell infiltrates were principally periductal and periislet, rather than the intraislet pattern characteristic of insulitis in unmanipulated NOD mice. Protection was not associated with the induction of "allogenic tolerance" or systemic autoimmunity. Accelerated IDDM occurs after injection of splenocytes from NOD donors into irradiated adult NOD recipients. When alpha/beta-TCR+ and alpha/beta-TCR- subsets of CD4-CD8- thymocytes were transferred with diabetogenic splenocytes and compared for their ability to prevent the development of IDDM in irradiated adult recipients, only the alpha/beta-TCR+ population was protective, confirming that NKT cells were responsible for this activity. The protective effect in the induced model of IDDM was neutralized by anti-IL-4 and anti-IL-10 monoclonal antibodies in vivo, indicating a role for at least one of these cytokines in NKT cell-mediated protection. These results have significant implications for the pathogenesis and potential prevention of IDDM in humans.

Glucose homeostasis in the nonobese diabetic mouse at the prediabetic stage.

Because few data were available on glucose homeostasis at the early prediabetic stage in the nonobese diabetic (NOD) mouse, we investigated glycemia, insulinemia, and pancreatic insulin content under basal conditions in both sexes of 4-, 6-, and 8-week-old fed NOD mice, compared with sex- and age-matched fed C57BL/6 mice. We also investigated glucose tolerance in both sexes of fasting 8-week-old NOD and C57BL/6 mice. The main results obtained under basal fed conditions, when comparing both strains, were lower glycemia and higher insulinemia in NOD females at all ages investigated and in NOD males (particularly at 6 weeks of age). Glucose tolerance tests showed that: 1) the blood glucose response to 1 g/kg i.p. glucose was less sustained in both sexes of 8-week-old NOD mice than in their control counterparts; 2) the blood insulin response to glucose (1 g/kg i.p.) appeared earlier in both sexes of NOD mice than in sex-matched C57BL/6 mice; 3) an unusual sexual dimorphism existed in NOD mice, compared with controls, with females secreting, in response to glucose, twice as much insulin as males; 4) dose-response studies (1-6 g/kg glucose) confirmed the lower increase in blood glucose levels in both sexes of NOD mice and their unusual sexual dimorphism in insulin secretion; and 5) glucose tolerance tests in 4- to 8-week-old NOD mice showed that although the sexual dimorphism in insulin secretion was not observed in 4-week-old mice, it was particularly striking at 6 weeks of age. Taken together, these results suggest that beta-cell hyperactivity exists in the NOD mouse at the early prediabetic stage, especially in NOD females.

Islet T Cells Secreting IFN-γ in NOD Mouse Diabetes: Arrest by p277 Peptide Treatment

Non-obese diabetic (NOD) mice spontaneously develop insulin-dependent (type 1) diabetes mellitus (IDDM) caused by T cells which destroy the insulin-producing islet β-cells. Since cytokines are involved in this auto-immune β-cell damage, we used an ELISPOT assay to enumerate the islet-associated T cells that secreted interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) or interleukin-4 (IL-4). We used mitogenic anti-CD3 antibody to activate all the T cells capable of responding, irrespective of their antigen specificity. We found that NOD females, more susceptible than males to IDDM, accumulated islet IFN-γ producers more rapidly with age than did the males. Acceleration of male IDDM by cyclophosphamide led to a marked increase in IFN-γ secreting islet T cells. In contrast, a decrease in IFN-γ-producing islet T cells was associated with arrest of IDDM by administration of peptide p277 of the 60kDa heat-shock protein (hsp60) to 12-week-old female NOD mice. The p277-treated mice later manifested a greater number of islets and fewer leukocytes per islet than did the mice treated with a bacterial hsp60 peptide. Thus, the development of diabetes could be correlated with the accumulation in the islets of T cells producing IFN-γ, and destructive insulitis could be downregulated by the administration of a single peptide.